Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane–bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase that is essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of the two domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to how the C-terminal lipase domain recognizes autophagic bodies to degrade them.     

Phospholamban of cardiac sarcoplasmic reticulum consists of two functionally distinct proteolipids

Phosphorylation of phospholamban by either a cAMP-dependent or a calmodulin-dependent kinase stimulates the Ca²⁺ transporting activity of cardiac sarcoplasmic reticulum membranes. It has now been found that phospholamban consists of 2 distinct proteins; one is the specific substrate for the cAMP-dependent phosphorylation, and the other for the calmodulin-dependent kinase. In spite of functional diversity, the 2 polypeptides share a number of properties. Among them, the proteolipid character, M_r, resistance to trypsinization, and subunit composition.     

The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers

Twinfilin is a ubiquitous and abundant actin monomer-binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K(D) = 0.05 microM) than ATP-G-actin (K(D) = 0.47 microM) under physiological ionic conditions and forms a relatively stable (k(off) = 1.8 s(-1)) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.     

CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with beta3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)-/- OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP-/- OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP-/- OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.     

Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function.

AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs.     

Image analysis reveals that Escherichia coli RecA protein consists of two domains.

The Escherichia coli RecA protein catalyzes homologous genetic recombination by forming helical polymers around DNA molecules. These polymers have an ATPase activity, which is essential for the movement of strands between two DNA molecules. One obstacle to structural studies of the RecA filament has been that the ATPase results in a dynamical polymer containing a mixture of states with respect to the bound ATP and its hydrolytic products. We have formed filaments which are trapped in the ADP-Pi state by substituting AIF4- for the Pi, and have used these stable filaments to generate a three-dimensional reconstruction from electron micrographs. The resolution of the reconstruction is sufficient to resolve the 38-k RecA subunit into two nearly equal domains. This reconstruction provides the most detailed view yet of the RecA protein, and serves as a framework within which existing biochemical data on RecA can be understood.     

Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit

The key step of N-glycosylation of proteins in the endoplasmic reticulum is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). It transfers the lipid-linked core-oligosaccharide to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains. Biochemical and genetic approaches have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1p, Swp1p, Stt3p, Ost1p, Ost2p, Ost4p, Ost5p, Ostp3 and Ost6p. By blue native polyacrylamide electrophoresis we show that yeast OST consists of two isoforms with distinct functions differing only in the presence of the two related Ost3 and Ost6p proteins. The OST6-complex was found to be important for cell wall integrity and temperature stress. Ost3p and Ost6p are not essential for OST activity, and can in part displace each other in the complex when overexpressed, suggesting a dynamic regulation of the complex formation.     

Sonication of mouse sperm membranes reveals distinct protein domains.

Molecular interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized. To begin to characterize sperm components that are involved in sperm-ZP interactions, we isolated and density fractionated sperm membranes. The membrane fractions recovered from a density fractionation protocol were characterized, and sonication was compared with vortexing for preparation of sperm membranes by examining the distribution of proteins in the membrane fractions obtained from these 2 protocols. Biochemical and microscopic analyses were used to determine the composition of the sonicated membrane fractions, and immunoblotting was used to identify fractions containing some of the previously suggested ZP3 receptors. Transmission electron microscopy revealed that bands 1-3 contained membrane vesicles and band 4 contained axonemal and midpiece fragments. SDS-PAGE revealed that bands 1 and 2 shared many proteins, but band 3 contained a number of unique proteins. Surface labeling with 125I demonstrated that bands 1 and 2 contained the majority of the sperm surface protein markers, whereas band 3 contained minor amounts of surface markers. Lectin-binding characteristics of sperm membrane glycoproteins were used to compare the relative distribution of glycosylated proteins in vortexed or sonicated membrane preparations. These characterizations indicate that sonication enhanced the differential distribution of sperm membrane proteins among the density fractions and suggests that this method is preferable for preparation of membrane fractions to be used for identification of proteins that mediate sperm-egg interactions.     

Syntaxin 8 has two functionally distinct di-leucine-based motifs

Syntaxin 8 has been shown to form the SNARE complex with syntaxin 7, vti1b and endobrevin. These have been shown to function as the machinery for the homotypic fusion of late endosomes. Recently, we showed that syntaxins 7 and 8 cycle through the plasma membrane, and that the di-leucine-based motifs in the cytoplasmic domain of syntaxins 7 and 8 respectively function in their endocytic and exocytic processes. However, we could not elucidate the mechanism by which syntaxin 8 cycles through the plasma membrane. In this study, we constructed several different syntaxin 8 molecules by mutating putative di-leucine-based motifs, and analyzed their intracellular localization and trafficking. We found a di-leucine-based motif in the cytoplasmic domain of syntaxin 8. It is similar to that of syntaxin 7, and functions in its endocytosis. These results suggest that in the cytoplasmic domain, syntaxin 8 has two functionally distinct di-leucine-based motifs that act independently in its endocytic and exocytic processes. This is the first report on two di-leucine-based motifs in the same molecule acting independently in distinct transport pathways.     

The amino-terminus of phytochrome A contains two distinct functional domains

The N-terminus of phytochrome A is important for the structural integrity and biological activity of the photoreceptor. Mutational analysis of the N-terminus by two different strategies created two distinct photoreceptors, one inactive and the other hyperactive when expressed in transgenic tobacco, suggesting that this region has multiple functional domains. To identify critical residues within this N-terminal region, a series of smaller deletions of oat phytochrome A were created, designated NB (Δ49–62), NC (Δ6–47), ND (Δ7–21), NE (Δ2–5), and NF (Δ6–12), and compared with a previously characterized deletion mutant NA (Δ7–69) and full-length oat phytochrome A. Using photochemical properties as a measure of chromoprotein structure, it was found that the region between residues 13 and 62 was important for the spectral integrity of the photoreceptor. These deletion mutants were also biologically inactive when expressed in both mature tobacco plants and seedlings grown under continuous far-red or red light. In contrast, deletion of the serine-rich region between residues 6 and 12 did not alter the photochemical properties but did produce a hyperactive photoreceptor, indicating this region may be involved in down-regulating phytochrome A activity. The data show that the N-terminus of phytochrome A contains two functional domains, one necessary for conformational stability and biological activity (residues 13–62), and the other involved in attenuating phytochrome responses (residues 6–12).     

The core protein of fibroblast proteoheparan sulphate consists of disulphide-bonded subunits.

Fibroblast proteoheparan sulphate has a disulphide-bonded subunit structure. The core protein appears to consist of two polypeptides each of Mr 80 000-100 000. As shown elsewhere [Carlstedt, Cöster, Malmström & Fransson (1983) J. Biol. Chem. in the press], both polypeptide molecules carry four to six heparan sulphate side chains (approx. Mr 20 000) and an unknown number of oligosaccharide units, giving the whole macromolecule an Mr in the range 300 000-400 000.